title: CYLD deubiquitinates RIP1 in the TNFα-induced necrosome to facilitate kinase activation and programmed necrosis.
abstract: BACKGROUND
  Necroptosis/programmed necrosis is initiated by a macro-molecular protein complex termed the necrosome. Receptor interacting protein kinase 1 (RIPK1/RIP1) and RIP3 are key components of the necrosome. TNFα is a prototypic inducer of necrosome activation, and it is widely believed that deubiquitination of RIP1 at the TNFR-1 signaling complex precedes transition of RIP1 into the cytosol where it forms the RIP1-RIP3 necrosome. Cylindromatosis (CYLD) is believed to promote programmed necrosis by facilitating RIP1 deubiquitination at this membrane receptor complex.
  
  
  RESULTS
  We demonstrate that RIP1 is indeed the primary target of CYLD in TNFα-induced programmed necrosis. We observed that CYLD does not regulate RIP1 ubiquitination at the TNF receptor. TNF and zVAD-induced programmed necrosis was highly attenuated in CYLD(-/-) cells. However, in the presence of cycloheximide or SMAC mimetics, programmed necrosis was only moderately reduced in CYLD(-/-) cells. Under the latter conditions, RIP1-RIP3 necrosome formation is only delayed, but not abolished in CYLD(-/-) cells. We further demonstrate that RIP1 within the NP-40 insoluble necrosome is ubiquitinated and that CYLD regulates RIP1 ubiquitination in this compartment. Hence, RIP1 ubiquitination in this late-forming complex is greatly increased in CYLD(-/-) cells. Increased RIP1 ubiquitination impairs RIP1 and RIP3 phosphorylation, a signature of kinase activation.
  
  
  CONCLUSIONS
  Our results show that CYLD regulates RIP1 ubiquitination in the TNFα-induced necrosome, but not in the TNFR-1 signaling complex. In cells sensitized to programmed necrosis with SMAC mimetics, CYLD is not essential for necrosome assembly. Since SMAC mimetics induces the loss of the E3 ligases cIAP1 and cIAP2, reduced RIP1 ubiquitination could lead to reduced requirement for CYLD to remove ubiquitin chains from RIP1 in the TNFR-1 complex. As increased RIP1 ubiquitination in the necrosome correlates with impaired RIP1 and RIP3 phosphorylation and function, these results suggest that CYLD controls RIP1 kinase activity during necrosome assembly.
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: David M
authors.initials: DM
authors.lastname: Moquin
authors.forename: Thomas
authors.initials: T
authors.lastname: McQuade
authors.forename: Francis Ka-Ming
authors.initials: FK
authors.lastname: Chan
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D000074749:Deubiquitinating Enzyme CYLD; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D009336:Necrosis / Q000235:genetics* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D047888:Receptors, Tumor Necrosis Factor, Type I; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D020690:GTPase-Activating Proteins; D009842:Oligopeptides; C490620:Ralbp1 protein, mouse; D047888:Receptors, Tumor Necrosis Factor, Type I; C532637:SMAC peptide; C506236:Tnfrsf1a protein, mouse; D014409:Tumor Necrosis Factor-alpha; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid; D003513:Cycloheximide; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse; C511601:CYLD protein, mouse; D000074749:Deubiquitinating Enzyme CYLD; D003546:Cysteine Endopeptidases